The re-emergence of tuberculosis has led to renewed interest in facultative intracellular pathogen, Mycobacterium tuberculosis. Soon after entering the host, the Mycobacterium is engulfed by macrophages. This encounter between bacterium and macrophage determines the outcome of the pathogen-host interaction. If bacteria are able to resist killing by the macrophage, they will remain in the host for decades, re-emerging to cause disease when the host immune system is compromised by old age, HIV infection, or chemotherapy. How mycobacterial pathogens are able to persist within the host for decades is unknown but it is believed they are in a non-replicative, dormant state. Our goal is to study the fate of Mycobacterium within the host cell, to characterize cellular and bacterial components required for bacterial entry into and survival within host cells, and to identify bacterial components required for persistence in the host. We are using M. marinum and M. ulcerans, both of which cause persistent disease in humans, as model systems for studying M. tuberculosis. We have shown that these two species are more closely related to M. tuberculosis than any mycobacterial pathogen aside from M. bovis. Our studies show that M. marinum, like M. tuberculosis, replicates and persists within macrophages. We have isolated a M. marinum derivative which has lost the ability to persist in host cells and are using this strain in complementation studies to identify components from M. marinum required for persistence and will determine whether they are present in M. tuberculosis as well. Many bacterial species contain a stationary phase sigma factor which turns on genes transcribed in dormant bacteria. We have identified a homologous sigma factor from M. tuberculosis, M. marinum, M. ulcerans, and M. haemophilum. Investigations are in progress to identify the role this gene may play in the ability of mycobacteria to persist within host cells. We found cloned mycobacterial DNA can be electroporated into M. marinum and that plasmid vectors which replicate in M. tuberculosis are stably maintained in M. marinum. M. marinum replicates much more rapidly than M. tuberculosis which makes this system invaluable in studying the genetics of mycobacterial virulence.